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Principle and application of the genome editing technology*(PDF)

Chinese Journal of Applied & Environmental Biology[ISSN:1006-687X/CN:51-1482/Q]

2016 02
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Principle and application of the genome editing technology*
HE Feiyan1 2 YAN Jianjun2 3 FENG Ruiyun2 ZHANG Aiping4 ZHANG Weifeng2 & BAI Yunfeng2 3**
1College of Bioengineering, Shanxi University, Taiyuan 030006, China2Institute of Crop Science, Shanxi Academy of Agricultural Sciences, Taiyuan 030006, China3China Key Laboratory of Loess Plateau Crop Gene Resources and Germplasm Creation, Ministry of
genome editing ZFNTALENCRISPR/Casprinciple molecular breeding

Elucidating gene function and improving biological status is one of the key research contents of biology. The useof gene targeting and transgenic technology is accompanied with problems including low efficiency, long time, safety doubtsand others. New genome editing techniques based on novel and efficient DNA targets include ZFN, TALEN and CRISPR/Cas. In this paper, we first review the three technology principles, which all based on DNA/DNA damage repair to achieveediting function, can perform target gene deletion, single nucleotide or polynucleotide fragments replacement, and targetgene modification in different species of the target gene. The new technology is simple, rapid, efficient and accurate. Then wecompare the three techniques in terms of composition, target recognition mode, editing features, technical difficulty and offtargeteffect. We also summarize the status and prospects of the application of this technology in crop genetics and breeding,livestock improvement and gene therapy. The review points out the shortcomings of the technology including off-target effectand construction problems, and provides with possible solutions. The application of ZFN, TALEN and CRISPR/Cas in themodel plant and grain crops is emphasized. At the end of this paper, we also introduce the present research situation and ourresearch ideology concerning the technology, proposing the establishment of the technology platform of genome editing basedon viral vector, which will make simpler and safer application of genome editing for plant genetic improvement.


1 谢科, 饶力群, 李红伟, 安学丽, 方才臣, 万向元. 基因组编辑技术在植物中的研究进展与应用前景[J]. 中国生物工程杂志, 2013, 33 (6):99-104 [Xie K, Rao LQ, Li HW, An XL, Fang CC, Wan XY. Researchprogress and application prospect of genome editing technology in plants[J]. Chin J Biol Eng, 2013, 33 (6): 99-104]2 程曦, 王文义, 邱金龙. 基因组编辑: 植物生物技术的机遇与挑战[J]. 生物技术通报, 2015, 31 (4): 25-33 [Cheng X, Wang WY, Qiu JL.Genome editing: opportunities and challenges in plant biotechnology [J].2015, 31 (4): 25-33]3 魏景亮, 吴添文, 阮进学, 牟玉莲. 基因组编辑技术改良家畜的研究进展[J]. 中国农业科技导报, 2014, 16 (1): 32-38 [Wei JL, Wu, TW,Ruan JX, Mou, YL. Research progress of improving livestock by genomeediting technology [J]. 2014, 16 (1): 32-38]4 Moscou MJ, Bogdanove AJ. A simple cipher governs DNA recognitionby TAL effectors [J]. Science, 2009, 326 (5959): 1501-15015 Sapranauskas R, Gasiunas G, Fremaux C, Barrangou R, HorvathP, Siksnys V. The Streptococcus thermophiles CRISPR/Cas systemprovides immunity in Escherichia coli [J]. Nucleic Acids Res, 2011, 39(21): 9275-92826 Lloyd A, Plaisier CL, Carroll D, Drews GN. Targeted mutagenesis usingzinc-finger nucleases in Arabidopsis [J]. Proc Natl Acad Sci USA, 2005,102: 2232-22377 Shukla VK, Doyon Y, Miller JC, DeKelver RC. Precise genomemodification in the crop species Zea maysusing zinc-finger nucleases[J].Nature, 2009, 459 (7245): 437-4418 Zhang F, Maeder ML, Unger-Wallace E, Hoshaw JP, Reyon D, ChristianM, Li X, Pierick CJ, Dobbs D, Peterson T, Joung JK, Voytas DF. Highfrequency targeted mutagenesis in Arabidopsis thaliana using zinc fingernucleases [J]. Proc Natl Acad Sci USA, 2010, 107: 12028-120339 Osakabe K, Osakabe Y, Toki S. Site-directed mutagenesis in Arabidopsisusing custom-designed zinc finger nucleases [J]. Proc Natl Acad Sci USA,2010, 107 (26): 12034-1203910 Curtin SJ, Zhang F, Sander JD, Haun WJ, Starker C, Baltes N, Reyon D,Dahlborg EJ, Goodwin MJ, Coffman AP, Dobbs D, Joung JK, Voytas DF,Stupar RM. Targeted mutagenesis of duplicated genes in soybean withzinc-finger nucleases [J]. Plant Physiol, 2011, 156 (2): 466-47311 Qi YP, Li XH, Zhang Y, Starker CG, Baltes NJ, Zhang F, Sander JD,Reyon D, Joung JK, Voytas DF. Targeted deletion and inversion oftandemly arrayed genes in Arabidopsis thaliana using zinc fingernucleases [J]. G3(Bethesda), 2013, 3 (10): 1707-1715.12 Cai CQ, Doyon Y, Ainley WM, Miller JC, Dekelver RC, Moehle EA.Targeted transgene integration in plant cells using designed zinc fingernucleases [J]. Plant Mol Biol, 2009, 69 (6): 699-70913 张金脉, 任兆瑞. TALENs: 一种新的基因定点修饰技术[J]. 生命科学,2013, 25 (12): 126-132 [Zhang JM, Ren ZR. TALENs: a novel gene sitedirected modification technique [J]. Life Sci, 2013, 25 (12): 126-132]14 Li T, Liu B, Spalding MH, Weeks DP, Yang B. High-efficiency TALENbasedgene editing produces disease-resistant rice [J].Nat Biotechnol,2012, 30: 390-39215 Shan QW, Wang YP, Chen KL, Liang,Z, Li J, Zhang Y, Zhang K, LiuJX, Voytas DF, Zheng XL, Zhang Y, Gao CX. Rapid and efficient genemodification in rice and Brachypodium using TALENs [J]. Mol Plant,2013, 6 (4): 1365-136816 Haun W, Coffman A, Clasen BM, Demorest ZL, Lowy A, Ray E,Retterath A, Stoddard T, Juillerat A, Cedrone F, Mathis L, Voytas DF,Zhang F. Improved soybean oil quality by targeted mutagenesis of thefatty acid desaturase 2 gene family [J]. Plant Biotechnol J, 2014, 12 (7):934-94017 Liang Z, Zhang K, Chen K, Gao C. Targeted mutagenesis in Zea maysusing TALENs and the CRISPR/Cas system [J]. J Genet Genom, 2014,41 (2): 63-6818 Wang Y, Cheng X, Shan Q, Zhang Y, Liu J, Gao C, Qiu J. Simultaneousediting of three homoeoalleles in hexaploid bread wheat confers heritableresistance to powdery mildew [J]. Nat Biotechnol, 2014, 32 (9): 947-95119 Li JF, Norville JE, Aach J, McCormack M, Zhang D, Bush J, ChurchGM, Sheen J. Multiplex and homologous recombination-mediatedgenome editing in Arabidopsis and Nicotiana benthamiana using guideRNA and Cas9 [J]. Nat Biotechnol, 2013, 31 (8): 688-69120 Shan Q, Wang Y, Li J, Zhang Y, Chen KL, Liang Z, Zhang K, Liu JX, XiJJ, Qiu JL, Gao CX. Targeted genome modification of crop plants using aCRISPR-Cas system [J]. Nat Biotechnol, 2013, 31 (8): 686-68821 Mao Y, Zhang H, Xu N, Zhang B, Gou F, Zhu JK. Application of theCRISPR-Cas system for efficient genome engineering in plants [J]. MolPlant, 2013, 6 (6): 2008-201122 Brooks C, Nekrasov V, Lippman ZB, Van Eck J. Efficient gene editingin tomato in the first generation using the clustered regularly interspacedshort palindromic repeats/CRISPR-associated9 system [J]. Plant Physiol,2014, 166 (3): 1292-129723 Sugano SS, Shirakawa M, Takagi J, Matsuda Y, Shimada T, Hara-Nishimura I, Kohchi T. CRISPR/Cas9 mediated targeted mutagenesis inthe liverwort Marchantia polymorpha L. [J]. Plant Cell Physiol, 2014, 55(3): 475-48124 Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B. Chen QJ. ACRISPR/Cas9 toolkit for multiplex genome editing in plants [J]. BMCPlant Biol, 2014, 14: 32725 Xie K, Minkenberg B, Yang Y. Boosting CRISPR/Cas9 multiplex editingcapability with the endogenous tRNA-processing system [J]. Proc NatlAcad Sci USA, 2015, 112 (11): 3570-357526 Liu W, Rudis MR, Peng Y, Mazarei M, Millwood RJ, Yang JP, XuW.Synthetic TAL effectors for targeted enhancement of transgeneexpression in plants [J]. Plant Biotechnol J, 2013, 12 (4): 436-44627 Ainley WM, Sastry-Dent L, Welter ME, Murray MG, Petolino JF. Traitstacking via targeted genome editing. [J]. Plant Biotechonl J, 2013, 11 (9):1126-113428 Even-Faitelson L, Samach A, Melamed-Bessudo C, Avivi-Ragolsk N,Levy A. Localized egg-cell expression of effector proteins for targetedmodification of the Arabidopsis genome[J]. Plant J, 2011, 68 (5):929-93729 Marton I, Zuker A, Shklarman E, Zeevi V, Tovkach A, Roffe S, Ovadis M,Tzfira T, Vainstein A. Nontransgenic genome modification in plant cells[J]. Plant Physiol, 2010, 154 (3): 1079-108730 Tovkach A, Zeevi V, Tzfira T, Tzfira T, Univ Michigan, Dept MolCellular, Dev Biol. A toolbox and procedural notes for characterizingnovel zinc finger nucleases for genome editing in plant cells [J]. Plant J,2009, 57 (4): 747-75731 Gurushidze M, Hensel G, Hiekel S, Schedel S, Valkov V, Kumlehn J.True-breeding targeted gene knock-out in barley using designer TALEnucleasein haploid cells [J]. PLoS ONE, 2014, 9 (3): e9204632 Christian M, Qi Y, Zhang Y, Voytas DF. Targeted mutagenesis ofArabidopsis thaliana using engineered TAL effector nucleases [J]. G3(Bethesda), 2013, 3 (10): 1697-170533 Wendt T, Holm P, Starker CG, Christian M, Voytas DF, Holme I. TALeffector nucleases induce mutations at a pre-selected location in thegenome of primary barley transformants [J]. Plant Mol Biol, 2013, 83 (3):279-28534 Feng Z, Mao Y, Xu N, Zhang BT, Wei PL, Yang DL, Wang Z, Zhang ZJ,Zheng R, Yang L, Zeng L, Liu XD, Zhu JK. Multigeneration analysisreveals the inheritance, specificity, and patterns of CRISPR/Cas-inducedgene modifications in Arabidopsis [J]. Proc Natl Acad Sci USA, 2014, 111(12): 4632-463735 Fauser F, Schml S, Puchta H. Both CRISPR/Cas-based nucleases andnickases can be used efficiently for genome engineering in Arabidopsisthaliana [J]. Plant J, 2014, 79 (2): 348-35936 Jiang W, Yang B, Weeks DP. Efficient CRISPR/Cas9-mediated geneediting in Arabidopsis thaliana and inheritance of modified genes in theT2 and T3 generations [J]. PLoS ONE, 2014, 9: e9922537 Zhou H, Liu B, Weeks DP, Spalding MH, Yang B. Large chromosomaldeletions and heritable small genetic changes induced by CRISPR/Cas9in rice [J]. Nucleic Acids Res, 2014, 42 (17): 10903-1091438 Zhang H, Zhang J, Wei P, Zhang B, Gou F, Feng Z, Mao Y, Yang L,Zhang H, Xu N, Zhu J. The CRISPR/Cas9 system produces specific andhomozygous targeted gene editing in rice in one generation [J]. PlantBiotechnol J, 2014, 12 (6): 797-80739 Feng Z, Zhang B, Ding W, Liu X, Yang D, Wei P, Cao F, Zhu S, ZhangF, Mao Y, Zhu J. Efficient genome editing in plants using a CRISPR/Cassystem [J]. Cell Res, 2013, 23 (10): 1229-123240 De Pater S, Neuteboom LW, Pinas JE. ZFN-induced mutagenesis andgene-targeting in Arabidopsis through Agrobacterium-mediated floraldip transformation [J]. Plant Biotechnol J, 2009, 7 (8): 821-83541 De Pater S, Pinas JE, Hooykaas PJ, van der Zaal BJ. ZFN-mediated genetargeting of the Arabidopsis protoporphyrinogen oxidase gene throughAgrobacterium-mediated floral dip transformation [J]. Plant Biotechnol J,2013, 11 (4): 510-51542 Baltes NJ, Gil-Humanes J, Cermak T, Atkins PA, Voytas DF. DNAreplicons for plant genome engineering [J]. Plant Cell, 2015, 26 (1): 151-16343 Wright DA, Townsend JA, Winfrey RJ, Irwin PA, Rajagopal J, Lonosky P.High-frequency homologous recombination in plants mediated by zincfingernucleases [J]. Plant J, 2005, 44 (4): 693-70544 Schiml S, Fauer F Holger P. The CRISPR/Cas system can be used asnuclease for in planta gene targeting and as paired nickases for directedmutagenesis in Arabidopsis resulting in heritable progeny [J]. Plant J,2014, 80 (6): 1139-115045 Gupta M, Dekelver RC, Palta A, Clifford C, Gopalan S, Miller JC, NovakS, Desloover D, Gachotte D, Connell J, Flook J, Patterson T, Robbins K,Rebar EJ, Gregory PD, Urnov FD. Petolino JF. Transcriptional activationof Brassica napus b-ketoacyl-ACP synthase II with an engineered zincfinger protein transcription factor [J]. Plant Biotechnol J, 2012, 10 (7):783-79146 Mahfouz MM, Li L,Piatek M, Fang XY, Mansour H, Bangarusamy DK,Zhu JK. Targeted transcriptional repression using a chimeric TALE–SRDX repressor protein [J]. Plant Mol Biol, 2012, 78 (3): 311-32147 Geissler R, Scholze H, Hahn S, Streubel J, Bonas U, Behrens SE, Boch,J. Transcriptional activators of human genes with programmable DNAspecificity[J]. PLoS ONE, 2011, 6 (5): e1950948 Piatek A, Ali Z, Baazim H, Baazim H, Li L, Abulfaraj A, ShareefSA, Aouida M. RNA - guided transcriptional regulation in planta viasynthetic dCas9 - based transcription factors [J]. Plant Biotechnol J,2014, 13 (4): 578-58949 Bibikova M, Golic M, Golic KG. Targeted chromosomal cleavage andmutagenesis in Drosophila using zinc-finger nucleases [J]. Genetics,2002, 161: 1169-117550 Takasu Y, Kobayashi I, Beumer K, Uchino K, Sezutsu H, Sajwan S,Carroll D, Tamura T, Zurovec M. Targeted mutagenesis in the silkwormBombyx mori using zinc finger nuclease mRNA injection [J]. InsectBiochem Mol Biol, 2010, 40: 759-76551 Ma SY, Zhang SL, Wang F, Liu YY, Xu HF, Liu C, Lin Y, Zhao P, XiaQY. Highly efficient and specific genome editing in silkworm usingcustom TALENs [J]. PLoS ONE, 2012, 7: e4503552 Watanabe T, Ochiai H, Sakuma T, Horch HW, Hamaguchi N, NakamuraT, Bando T, Mito, T. Non-transgenic genome modifications in ahemimetabolous insect using zinc-finger and TAL effector nucleases [J].Nat Commun, 2012, 3: 101753 Liu J, Li C, Yu Z, Huang P, Wu H, Wei C, Zhu N, Shen Y, Chen Y,Zhang B, Deng W. Efficient and specific modifications of the Drosophilagenome by means of an easy TALEN strategy [J]. J Genet Genomics,2012, 39: 209-21554 Gratz SJ, Cummings AM, Nguyen JN, Hamm DC, Donohu Lk. Genomeengineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease[J]. Genetics, 2013, 194: 1029-103555 张圣棂. TALEN介导的家蚕基因组编辑研究[D]. 重庆: 西南大学,2015 [Zhang SL. Study on TALEN mediated genome editing in silkworm[D]. Chongqing, Southwestern University, 2015]56 Yu S, Luo J, Song Z, Ding F, Dai Y, LI N. Hightly efficient modificationof beta-lactoglobulin (BLG) gene via zinc-finger nuclease in cattle [J].Cell Res, 2011, 21 (11): 1638-164057 Hauschild J, Petersen B, Santiago Y, Queisser AL, Carnwath JW, HahnAL, Zhang L, Meng X, Gregory PD, Schwinzer R, Cost GJ, NiemannH. Efficient generation of a biallelic knockout in pigs using zinc-fingernucleases [J]. Proc Natl Acad Sci USA, 2011, 108 (29): 12013-1201758 Carlson DF, Tan W, Lillico SG. Eifficient TAlEN-mediated geneknockout in livestock [J]. Proc Natl Acad Sci USA, 2012, 109 (43): 17382-1738759 Perez EE, Wang J, Miller JC, Jouvenot Y, Kim KA, Liu O, Wang N, Lee G,Bartsevich VV, Lee YL, Guschin DY, Rupniewski I, Waite AJ, CarpenitoC, Carroll RG ,Orange JS, Urnov FD, Rebar EJ, Ando D, Gregory PD,Riley JL, Holmes MC, June CH. Establishment of HIV-1 resistancein CD4 T cell by genome editing using zinc-finger nucjeases [J]. NatBiotechnol, 2008, 26 (7): 808-81660 Holt N, Wang J, Kim K, Hriedman G, Wang X, Taupin V, Cannon PM.Human hematopoietic stem/progenitor cells modified by zinc-fingernucleases targeted to CCR5 contril HIV-1 in viro [J]. Nat Biotechnol,2010, 28 (8): 839-84761 Mussolino C, Morbizer R, Lutge F, Dannemann N, Lahaye T, Cathomen T.A novel TALE nuclease scaffold enables high genome editing activity incombination with low toxicity [J]. Nat Methods, 2012, 9 (8): 805-80762 Sebastiano V, Maeder ML, Angstman JF, Haddad B, Khayter C, Yeo DTGoodwin MJ, Hawkins J, Ramirez CL, Batista LFZ, Artandi SE, WernigM, Joung JK. In situ genetic correction of the sickle cell anemia mutationin human induced pluripoptent stem cells using engineered zine fingernucleases [J]. Stem Cells, 2011, 29 (11): 1717-172663 An MC, Zhang N, Scott G, Montoro D, Wittkop T, Mooney S, Melov S,Ellerby LM. Genetic correction of huntingtons disease phenotypes ininduced pluripotent stem cells [J]. Stem Cells, 2012, 11 (2): 253-26364 Greenwald DL, Cashm an SM, Kumar-Singh R. Engineered zinc fingernuclease-mediated homologous recombination of the human rhodopsingene [J]. Invest Ophthalmol Vis Sci, 2010, 51 (12): 6374-638065 张然, 田浤, 高向东, 姚文兵. 新一代基因组编辑技术在基因治疗及生物制药领域中的应用[J]. 中国药科大学学报, 2014, 45 (3): 501-510[Zhang R, Tian H, Gao XD, Yao WB. Application of new generationgenome editing technology in gene therapy and biological pharmacy [J].J China Med Univ, 2014, 45 (3): 501-510]


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